CN110300603B

CN110300603B - CD47-CAR-T cells

Info

Publication number: CN110300603B
Application number: CN201880012017.5A
Authority: CN
Inventors: 吴力军; V·戈卢鲍夫斯卡亚
Original assignee: Genxi Biotechnology Shanghai Co ltd
Current assignee: Genxi Biotechnology Shanghai Co ltd
Priority date: 2017-02-14
Filing date: 2018-02-09
Publication date: 2023-04-14
Anticipated expiration: 2038-02-09
Also published as: US11692034B2; CN110300603A; WO2018152033A1; US20190338028A1

Abstract

The present invention provides a Chimeric Antigen Receptor (CAR) fusion protein comprising, from N-terminus to C-terminus: (i) Comprising V _H And V _L The single chain variable fragment (scFv) of (i), wherein the scFv has activity against CD47, (ii) a transmembrane domain, (iii) at least one costimulatory domain, and (iv) an activation domain. In one embodiment, the scFv is derived from a humanized anti-CD 47 antibody. The invention also provides T cells modified to express a CAR of the invention.

Description

CD47-CAR-T cells

Sequence listing, table, or computer program reference

The sequence list is submitted simultaneously through EFS-Web in an ASCII format text file, the file name is 'sequence list txt', the creation date is 2018, 2,8 and the size is 26.1 kilobytes. The sequence listing submitted by EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to humanized CD47-CAR-T cells that efficiently attack tumor cells that overexpress the CD47 tumor antigen.

Background

Immunotherapy is becoming a very promising approach to cancer treatment. T cells or T lymphocytes are the armed force of our immune system, which constantly looks for foreign antigens and distinguishes abnormal (cancerous or infected) from normal cells. Genetic modification of T cells with CARs is a common approach to designing tumor-specific T cells. CAR-T cells targeting tumor-associated antigens can be infused into patients (known as adoptive T cell therapy), representing an effective immunotherapeutic approach. An advantage of CAR-T technology over chemotherapy or antibodies is that reprogrammed engineered T cells can proliferate and persist in a patient, acting like a live drug.

Generally, CARs (chimeric antigen receptors) include monoclonal antibody-derived single-chain variable fragments (scfvs) linked by a hinge and a transmembrane domain to a variable number of intracellular signaling domains and a single intracellular activating CD3-zeta domain.

Figure 1 shows the evolution of CARs from first generation (left, without costimulatory domain) to second generation (middle, with one costimulatory domain CD28 or 4-BB) to third generation (with two or more costimulatory domains), see Golubovskaya, wu, cancer, 2016, 3/15; 8 (3). The generation of CARs with multiple co-stimulatory domains (third generation CARs) resulted in increased cytolytic activity and significantly improved the persistence of CAR-T cells, exhibiting enhanced anti-tumor activity.

CD47 is a cell surface glycoprotein of the immunoglobulin superfamily that is frequently overexpressed in hematological tumors (leukemias, lymphomas, see Chao et al, cells, 2010,142,699-713, and multiple myeloma) and solid tumors such as ovarian cancer, small cell lung cancer, pancreatic glands, gliomas, glioblastoma, pediatric brain tumors, and other types of tumors (Chao, supra, and Weiskopf, j. CD47 is also considered to be "don't eat my signal" by binding SIRP-alpha (signal transduction regulatory protein alpha) and blocking SIRP-mediated phagocytosis of tumor cells (Weiskopf, supra). Studies have shown that high CD47 expression is associated with poor clinical outcome in some tumor patients and is considered a clinical prognostic factor. Such tumors include hematological tumors, such as non-hodgkin lymphoma (Chao, supra) or acute myeloid leukemia (Majeti et al, cell 2009,138, 286-299), as well as solid tumors, such as ovarian cancer, glioblastoma, glioma, etc. (Willingham et al, proc.natl.acad.sci.usa 2012,109, 6662-6667). In addition, CD47 signaling has been shown to play a key role in the maintenance of tumor initiating cells or cancer stem cells (Kaur et al, oncotarget,2016,7,10133-10152). CD47 is highly expressed in cancer stem cells, the most aggressive tumor cell type (Cioffi et al, clinical cancer research, 2015,21,2325-2337). Using CD47, the interaction of CD47 and SIRP alpha on macrophages was blocked, inducing phagocytosis of CD 47-positive tumors (Kim et al, leukemia, 2012,26,2538-2545).

There is a need for an improved adoptive T cell immunotherapy with improved therapeutic efficacy and reduced toxicity.

Brief description of the drawings

FIG. 1 shows the structure of a first to third generation CAR.

Figure 2 shows the structure of the CD47CAR construct.

FIG. 3A shows cytotoxicity of CD47-CAR-T cells versus T cells and Mock-CAR-T cells. Quantitation of RTCA from three independent experiments is shown. * Two-way analysis of variance (2-way ANOVA) and Tukey's late test for p <0.0001CD47-CAR-T cells versus T cells and Mock CAR-T cells.

FIG. 3B shows that CD47-CAR-T cells produce IL-2 in a CD 47-dependent manner; high in CD47 positive cells and low in CD47 negative cells. Effective target ratio E: t is 1:1. the bar graph shows the average of IL-2 secretion from CD47CAR-T cells in two independent experiments. * p <0.05, SKOV-3 and A375 cells versus student's T-test on T-cells, mock CAR-T cells, A549 cells and Hep3B cells.

Figure 4A shows that CD47 expression is significantly higher than CD24 expression in BxPC3 pancreatic cancer cells. Bar graphs show the mean ratio of MFI of CD24 and CD47 expressed by BxPC3 cells versus isotype control IgG1 ± standard deviation from two independent experiments. * p =0.029.

FIG. 4B shows that CD47-CAR-T cells efficiently kill BxPC3 cancer cell line in the RTCA assay.

Figure 4C shows that CD47-CAR-T cells secrete high levels of cytokines against BxPC3 cells in vitro: IFN- γ, IL-2 and IL-6, consistent with the high cytotoxicity of CD47-CAR-T cells on BxPCR3 cells as shown in FIG. 4B. The supernatant from the RTCA assay was used for the assay of the cytokine ILISA. E: t =10:1.* p <0.05.

Figure 4D shows that CD47-CAR-T cells significantly reduced tumor growth of BxPC3, p =0.006, cd47-CAR-T cells compared to 1xPBS control. n =4-5 mice, CD47/CD24-CAR-T cells and 1xPBS groups, respectively.

Figure 4E shows that CAR-T cells did not affect mouse body weight in CD47-CAR-T cells, CD24-CAR-T cells, and 1xPBS control. Mouse body weight was measured in grams twice a week.

Fig. 4F and 4G show that CD47CAR-T cells significantly reduced tumor size and weight, respectively. p <0.05, CD47-CAR-T cells compared to CD24-CAR-T cell control and 1xPBS group. Mice tumors were analyzed for imaging (4F) and weight was measured in grams (4G).

FIG. 5A shows the amino acid sequence of a humanized CD47ScFv. The upper diagram: CD47ScFv of the mouse B6H12 antibody. The following figures: humanized CD47.

Fig. 5B shows that ELISA demonstrated high binding activity of humanized CD47ScFv to CD47 antigen. Human PDL-1 antigen was used as a negative control. The binding activity is significantly higher than that of the mouse CD47ScFv. The bar graph shows the average binding activity from two independent experiments. * p =0.0025, mouse CD47 versus PDL-1 control; * P =0.0025, humanized CD47 compared to PDL-1 control; and p =0.0028, humanized CD47 compared to mouse CD47.

FIG. 5C shows IHC staining of tumor and normal samples with humanized and mouse CD47 ScFvs. Similar staining was observed. IHC of humanized CD47ScFv showed high staining in tumor samples and low staining in normal tissues.

Figure 6A shows RTCA cytotoxicity assays with humanized CD47-CAR-T cells, and CD47 positive or CD47 negative Hela-CD19 tumor target cells. The CD47-CAR-T cells were cytotoxic to CD47 positive Hela-CD19 cells but not to CD47 negative Hela-CD19 cells. The positive control CD19-CAR-T cells effectively killed CD19 positive Hela-CD19 cells.

Figure 6B illustrates the quantitative results of the RTCA assay, which shows that CD47-CAR-T cells have significantly enhanced cytotoxicity against CD 47-positive cancer cell lines, but not against CD 47-negative Hela-CD19 cells. * p <0.001,CD47-CAR-T cells compared to Mock-CAR-T cells.

Figure 6C shows IL-2 secretion by humanized CD47-CAR-T cells against CD47 positive SKOV-3 cells. E: t ratio =1:1.p <0.05, cd47-CAR-T cells compared to control, n =3.

Figure 6D shows that CD47-CAR-T cells do not secrete IL-2 against CD 47-negative Hela-CD19 cells. Mock-CAR-T cells are negative control cells. CD19-CAR-T cells are positive control cells against Hela-CD19 target cells. E: t ratio =10:1.p <0.05, cd19-CAR-T cells compared to control, n =3.

Figure 7 shows bispecific EGFR-CD47CAR-T cells kill EGFR-negative MCF-7 cells.

Figures 8A and 8B show that bispecific EGFR-CD47CAR-T cells effectively kill EGFR-positive and CD 47-positive BxPC3 pancreatic cancer cells (figure 8A) and SKOV ovarian cancer cells (figure 8B).

Detailed Description

Definition of

As used herein, "adoptive cell therapy" (ACT) is a treatment using cancer patients' own T lymphocytes with anti-tumor activity, which are expanded in vitro and re-infused into patients with cancer.

As used herein, "affinity" is the strength of binding of a single molecule to its ligand. Usually by balancing the dissociation constant (K) _D Or Kd) measurement and reporting of affinity, which is used to assess and rank the strength of bimolecular interactions.

As used herein, a "Chimeric Antigen Receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are also referred to as "chimeric receptors", "T-bodies" or "Chimeric Immunoreceptors (CIRs)". The term "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding an antigen. "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit biological processes in a cell.

As used herein, "domain" or "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.

As used herein, a FLAG tag, or FLAG octapeptide, or FLAG epitope refers to a polypeptide protein tag that can be added to a protein using recombinant DNA techniques, having a motif with the sequence DYKDDDD (SEQ ID No.: 1). It may be fused to the C-terminus or N-terminus of the protein, or inserted into the protein.

As used herein, the term "humanization" generally refers to the process of using genetic engineering techniques to render antibodies or immunoglobulin-binding proteins and polypeptides derived from non-human species (e.g., mice or rats) less immunogenic to humans while still retaining the antigen-binding properties of the original antibody. In some embodiments, the binding domain of an antibody or immunoglobulin (e.g., light and heavy chain variable regions, fab, scFv) that binds the protein or polypeptide is humanized. Non-human binding domains can be humanized, including remodeling, hyperchimerism, and veneering, using a technique known as CDR grafting. Other regions of antibodies or immunoglobulins that bind proteins and polypeptides, such as the hinge and constant region domains, may also be humanized if derived from non-human sources.

As used herein, "single chain variable fragment (scFv)" refers to a single chain polypeptide derived from an antibody that retains the ability to bind antigen. Examples of scfvs include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods for making scFv are well known to those skilled in the art.

As used herein, "tumor antigen" refers to an antigenic biomolecule, the expression of which results in cancer.

Description of the preferred embodiment

The present invention provides CD 47-CARs and CD47-CAR-T cells that target the CD47 tumor antigen, with CD47 being highly overexpressed in many types of cancer, such as ovarian cancer, bladder cancer, leukemia, and lymphoma. The present invention contemplates CAR (chimeric antigen receptor) -T cells that bind to the CD47 antigen. The present invention utilizes ScFv (single chain variable fragments) from mouse CD47 antibodies and humanized CD47 antibodies to generate CD47-CAR-T cells for targeting different cancer cell lines. The CD47-CAR-T cells of the invention are highly cytotoxic to several cancer cells (pancreatic cancer, ovarian cancer cell lines). The advantage of the humanized scFv is that it reduces the potential immune response of the patient to the mouse sequence.

The present invention relates to a Chimeric Antigen Receptor (CAR) fusion protein comprising, from N-terminus to C-terminus: (i) Comprising V _H And V _L The single chain variable fragment (scFv) of (i), wherein the scFv has a high affinity for CD47, (ii) a transmembrane domain, (iii) a CD28 costimulatory domain, and (iv) an activation domain. The CAR of (i) may further comprise a second scFv, wherein the second scFv is directed against another tumor antigen, such as Epidermal Growth Factor Receptor (EGFR); such CARs are referred to as bispecific CARs.

The CAR construct (figure 2) comprises a CD8 signal peptide, CD47scFv, CD8 hinge, CD28 transmembrane domain, and CD3 zeta activation domain.

The CAR of the invention comprises a single chain variable fragment (scFv) that specifically binds to human CD47. The heavy (H chain) and light (L chain) chain fragments of the anti-CD 47 antibody are linked by a linker sequence. For example, the linker may be 5-20 amino acids. The scFv structure may be VL-linker-VH or VH-linker-VL from N-to C-terminus.

The CAR of the invention comprises a transmembrane domain. The transmembrane domain may be derived from a native polypeptide or may be artificially designed. The transmembrane domain derived from a native polypeptide may be obtained from any membrane-bound or transmembrane protein. For example, the transmembrane domain of the T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD 3-epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR may be used. Artificially designed transmembrane domains are polypeptides that contain predominantly hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is included at each end of the synthetic transmembrane domain. In preferred embodiments, the transmembrane domain is derived from CD28 or CD8, which provides good receptor stability.

In the present invention, the co-stimulatory domain is selected from the group consisting of: human CD28, 4-1BB (CD 137), ICOS-1, CD27, OX 40 (CD 137), DAP10, and GITR (AITR).

The intracellular domain (activation domain) is the signaling part of the CAR. Upon recognition of the antigen, the receptor aggregates and transmits a signal to the cell. The most commonly used intracellular domain module is CD3-zeta (CD 3Z or CD3 zeta), which contains 3 ITAMs. Which upon antigen binding transmits an activation signal to the T cell. CD3-zeta does not provide a fully effective activation signal and requires additional costimulatory signaling. For example, one or more costimulatory domains can be used with CD3-zeta to deliver a proliferation/survival signal.

Optionally, the CAR fusion protein is comprised at the N-terminus of the scFv, or the C-terminus of the scFv, or the V _H And V _L FLAG tag in between. FLAG tag needs to be localized in cellThe ectodomain, rather than the intracellular domain. In addition to the FLAG tag, other tags may be used in the construct. The FLAG tag is a preferred tag because it does not cause immunogenicity and can reduce cytokine secretion levels.

The CAR of the invention may comprise a signal peptide linked to the N-terminus of an ScFv such that when the CAR is expressed intracellularly, for example in a T cell, the nascent protein is directed upon expression to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may comprise a long stretch of hydrophobic amino acids that tend to form a single alpha-helix. The signal peptide may start with a short positively charged amino acid stretch, which helps to enforce the proper topology of the polypeptide during translocation. At the end of the signal peptide, there is usually a stretch of amino acids recognized and cleaved by the signal peptidase. Signal peptidases can cleave during or after translocation is complete to produce free signal peptide and mature protein. Subsequently, the free signal peptide is digested by specific proteases. For example, the signal peptide may be derived from human CD8 or GM-CSF, or mutants thereof having 1 or 2 amino acid mutations, provided that the signal peptide still functions to direct cell surface expression of the CAR.

The CAR of the invention may comprise a spacer sequence that acts as a hinge to connect the scFv to the transmembrane domain and spatially separate the antigen binding domain from the intracellular domain. The flexible spacer allows the binding domains to be oriented in different directions to enable binding to the tumor antigen. The spacer sequence may comprise, for example, an IgG1Fc region, an IgG1 hinge, or a CD8 stem loop, or a combination thereof. Human CD28 or CD8 stem loops are preferred.

The invention provides a nucleic acid encoding the CAR described above. Nucleic acids encoding the CAR can be prepared by conventional methods, utilizing the amino acid sequence of the particular CAR. The nucleotide sequence encoding the amino acid sequence can be obtained using the NCBI RefSeq ID or GenBenk accession number of the amino acid sequence of each of the aforementioned domains. The nucleic acids of the invention can be prepared using standard molecular biology and/or chemistry methods. For example, a nucleic acid can be synthesized based on a base sequence, and the nucleic acid of the present invention can be prepared by fusing DNA fragments obtained from a cDNA library using Polymerase Chain Reaction (PCR).

The nucleic acid encoding the CAR of the invention can be inserted into a vector, and the vector can be introduced into a cell. For example, viral vectors such as retroviral vectors (including oncogenic retroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian viral vectors, vaccinia viral vectors or sendai viral vectors, epstein-barr virus (EBV) vectors, and HSV vectors can be used. As the viral vector, it is preferable to use a viral vector lacking the replication ability so as not to self-replicate in the infected cell.

For example, when a retroviral vector is used, the method of the present invention may be carried out by selecting an appropriate packaging cell based on the LTR sequence and packaging signal sequence possessed by the vector, and preparing a retroviral particle using the packaging cell. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP + E-86, GP + envAm-12 and Psi-Crip. Also 293 cells or 293T cells with high transfection efficiency can be used for the preparation of retroviral particles. A variety of retroviral vectors based on the production of retroviruses and packaging cells are widely available from a number of companies.

The invention provides T cells modified to express a chimeric antigen receptor fusion protein as described above. The CAR-T cells of the invention bind to human CD47 via CAR, thereby transmitting a signal into the cell, which in turn activates the cell. Activation of the CAR-expressing cell may vary depending on the kind of host cell and the intracellular domain of the CAR, and may be confirmed based on, for example, release of cytokines, improvement of cell proliferation rate, change of cell surface molecules, and the like as indicators.

T cells modified to express the CAR can be used as a therapeutic agent for a disease. The therapeutic agent comprises CAR-expressing T cells as an active ingredient, and may also comprise a suitable excipient. Examples of excipients include pharmaceutically acceptable excipients known to those skilled in the art.

The invention demonstrates the high activity of CD47-CAR-T cells against highly expressed CD47 cancer cell lines. The CD47-CAR-T cells of the invention effectively kill ovarian, pancreatic and other cancer cells and produce high levels of cytokines associated with CD47 antigen expression. The present invention demonstrates that intratumoral injection of CD47-CAR-T cells significantly reduces tumor growth of pancreatic BxPC3 xenografts compared to two controls (1 xPBS and CD24-CAR-T cells); at the same time, there was increased human CD3 ζ IHC staining in CD47-CAR-T cell treated tumors compared to control tumors.

The inventors designed a humanized CD47ScFv that binds human CD47 antigen, verified by ELISA and IHC staining of human tumor samples. Flanking the mouse CDRs, the humanized CD47scFV replaced the mouse framework sequence with a human framework sequence, thus reducing the potential immune response against the mouse scFV. The humanized CD47 antibody has some mutations in the mouse CDRs which increase binding to the CD47 antigen.

The humanized CD47-CAR-T cells of the invention effectively kill cancer cell lines with high expression of CD47, do not kill CD47 negative cancer cells and do not produce cytokines. Thus, mouse CD47-CAR-T cells and humanized CD47-CAR-T cells provide a cell therapy for solid tumors and hematologic cancers.

The present application shows that CD47-CAR-T cells are highly specific for CD47 positive cancer cells with high expression of CD47, while lacking CAR-T activity in target cells with low expression of CD47, such as Hela-CD19 cells. Thus, there is a therapeutic window for CD47-CAR-T cells to affect only CD 47-high expressing cells. Furthermore, CD47-CAR-T cells did not reduce mouse body weight, indicating that CAR-T cells have no toxic effects. Different methods of modulating CAR-T cells can be applied, such as on-off mechanisms; a bispecific CAR; to the modulation of cytokines of cytokine release syndrome, as well as other methods to increase safety and overcome the major challenges of CAR-T cell therapy.

Intratumoral injection of CD47-CAR-T cells increases the safety of these cells. Most CD47-CAR-T cells are localized within the tumor rather than in the blood. Therefore, regional intratumoral delivery of CD47-CAR-T cells is a clinically viable approach. Regional delivery of CD47-CAR-T cells may be beneficial for improved safety, with less inhibition of microenvironment and other mechanisms than direct delivery of CAR-T to tumors.

CD47 is also highly expressed in cancer stem cells, which are the most aggressive tumor cell type. Thus, CD47-CAR-T cells can be used to target cancer stem cells.

The present invention demonstrates a method of targeting cancer cells with CD47-CAR-T cells. Demonstrates the high efficiency of CD47-CAR-T cells against cancer cells in vitro and in vivo, and provides anti-cancer cell therapy.

Bispecific CAR-T cells targeting CD47 and another tumor antigen (EGFR, HER-2, VEGFR, etc.) can be used for immunotherapy. The bispecific CAR-T cell construct contains a first scFv to CD47 and a second scFv to a second tumor antigen. CAR-T cells with bispecific antibodies can target cancer cells that overexpress both tumor antigens more efficiently and specifically than CAR-T cells with a single antibody. When one tumor antigen is down-regulated, bispecific CAR-T cells may be advantageous because bispecific CAR-T cells can target another tumor antigen. The EGFR scFv is illustrated in example 14 of the present application. Sadelain et al reported HER-2scFv (J.Immunol.2009, 183: 5563-5574), chinnanaamy et al reported VEGFR scFv (J.Clin.invest.2010, 120 (11): 3953-3968); the entire contents of which are incorporated herein by reference.

The combination of CD47-CAR-T with CAR-T targeting other tumor antigens or tumor microenvironment antigens (VEGFR-1-3) (dual CAR-T) can be used to enhance the activity of CD47-CAR monotherapy.

CD47CAR-T can be used in combination with different chemotherapies: (ii) a checkpoint inhibitor; targeted therapies, small molecule inhibitors and antibodies.

Tag (FLAG tag or other tag) conjugated CD47scFv can be used in CAR constructs.

For the same CD47-scFv within a CAR, a third generation CAR-T or other co-activation signaling domain can be used.

CD47-CAR-T cells can be used to activate phagocytosis and block "don' T eat me" signaling.

The following examples further illustrate the invention. The following examples are intended to illustrate the invention and should not be construed as limiting.

Examples

Materials and methods

EXAMPLE 1 cell lines

HeLa cells were purchased from ATCC (Manassas, va.) and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin. Hela-CD19 cells stably expressing CD19 were maintained in DMEM containing 10% fbs, puromycin and penicillin/streptomycin as described in Berahovich et al, front.biosci. (edited by Landmark) 2017,22,1644-1654. Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from whole blood obtained from the Stanford Hospital blood center (Stanford, CA, USA) using Ficoll-Paque solution (GE healthcare, chicago, IL) according to the protocol approved by IRB. HEK293FT cells from AlStem (Ristonem, CA, USA) were cultured in DMEM containing 10% FBS and penicillin/streptomycin. SKOV-3 cell lines were obtained from ATCC and cultured in RPMI supplemented with 10% FBS and penicillin/streptomycin. Normal human keratinocytes were obtained from Lonza corporation and cultured in keratinocyte medium (lomsa, anaheim, CA) according to the manufacturer's protocol. BxPC3, PANC-1, A1847, A375, A549 and Hep-3B were obtained from ATCC and cultured in DMEM containing 10% FBS and penicillin/streptomycin. Cell lines were flow cytometrically verified in our laboratory using cell-specific surface markers, or verified by ATCC.

Example 2CAR constructs

The CD47ScFv from the B6H12 antibody was inserted between the CD8- α signal peptide and the CD8- α hinge using Nhe I and Xho I, downstream fused to the CD28 transmembrane domain, CD28 coactivation domain, and CD3 activation domain. This CD47-CAR construct was flanked by Xba I and EcoR I sites in a pCD510 lentiviral vector (systematic bioscience, palo alto, CA). The same construction was performed with humanized CD47ScFv and Mock control ScFv (intracellular protein), which were referred to as humanized CD47 and Mock-CAR, respectively. As described above, CD24ScFv, CD24 (clone 4F4E 10) antibody from Promab biotechnology (limekan, CA) was used for the CD24-CAR construct. CAR was synthesized by Syno bioscience (beijing, china) and sequenced in both directions.

Example 3 Generation of CAR-encoding lentiviruses

293FT cells, lentivirus packaging mix, as described by Berahovich et alCompound and transfection agent (Alstem, richarden, CA), lentiviral CAR was used to generate lentiviruses. The virus titers were determined by quantitative RT-PCR using a Lenti-X qRT-PCR kit (Takara biology, mountain City, CA) and a 7900HT thermocycler (Semmerfel technology, south old Jinshan, CA) according to the manufacturer's protocol. Lentiviral titers were expressed as pfu/mL, ranging from 1-10X 10 ⁸ pfu/mL。

Example 4 Generation and expansion of CAR-T cells

PBMC were processed at 1X 10 ⁶ cells/mL were suspended in AIM V-Albumax medium (Sammerfeill) containing 10% FBS and 300U/mL IL-2 (Sammerfeill). PBMCs were activated with the same number of CD3/CD28 immunomagnetic beads (sequoyield) and cultured in untreated 24-well plates. At 24 and 48 hours, lentivirus with a multiplicity of infection (MOI) of 5 was added to the culture with 1. Mu.L of TransPlus transduction enhancer (AlStem). Counting CAR-T cells every 2-3 days, adding fresh medium containing 300U/mL IL-2 to the culture to maintain the cell density at 1X 10 ⁶ cells/mL.

Example 5 flow cytometry

To detect expression of CAR, 5X 10 ⁵ Cells were suspended in 100. Mu.L of buffer (1 XPBS containing 0.5% BSA) and incubated on ice for 10 min with 1. Mu.L of human serum (Jackson Immunity research, west Grov, pa., USA). Subsequently, 1. Mu.L of Allophycocyanin (APC) -labeled anti-CD 3 (eBioscience, san Diego, CA, USA), 2. Mu.L of 7-amino actinomycin D (7-AAD, bioLegend, san Diego, CA, USA), and 2. Mu.L of anti-F (ab) 2 or its isotype control were added, and the cells were incubated on ice for 30 minutes. Cells were washed with buffer and harvested on a FACSCalibur (BD bioscience, san jose, CA). Cells were first analyzed for light scattering of 7-AAD staining and then (plotted) 7-AAD was labeled ^- Live-gated cells (live gated cells) were used for CD3 staining with F (ab) 2 staining or isotype control staining. In some experiments, only anti-F (ab) 2 staining was performed. For mouse tumor studies, 100. Mu.l of blood was stained with 1. Mu.l of

APC anti-CD

3, 2. Mu.l of Fluorescein Isothiocyanate (FITC) -labeled anti-CD 8a (eBioscience), 2. Mu.l of 7-AAD for 30 minutes at room temperature. 3.5mL of RBC lysate (150 mM NH) ₄ Cl，10mM NaHCO ₃ 1mM EDTA pH 8), then white blood cells were collected by centrifugation and washed with 2mL of cold buffer prior to harvest. To express CD47, mouse B6H12 antibody from BioLegend was used at a concentration of 10 μ g/mL according to the manufacturer's protocol. To express CD24, mouse CD24 clone 4F4E10 (Promab Biotechnology, rismann, calif., USA) was used at 10. Mu.g/mL. Isotype IgG1 isotype antibody from BioLegend was used at 10. Mu.g/mL.

Example 6 real-time cytotoxicity assay (RTCA)

Adherent target cells were added at 1X 10 per well ⁴ Cells were seeded into 96-well E-plates (Acea biosciences, san diego, CA, USA) and monitored overnight in culture using the impedance-based real-time cell assay (RTCA) icellegence system (Acea biosciences). The next day, the medium was removed and the total content of 10% FBS. + -. 1X 10 ⁵ AIM V-AlbuMAX medium replacement of effector cells (CAR-T cells or untransduced T cells) in triplicate. Cells were monitored for an additional 2 days with the RTCA system and impedance was plotted over time. The cell lysis was calculated as: (target cell impedance of non-effector cell-target cell impedance of effector cell) x 100/target cell impedance of non-effector cell.

Example 7 cytokine ELISA assay

In a U-bottom 96-well plate, target cells and effector cells (CAR-T cells or untransduced T cells) were mixed at a ratio of 1: ratio of 1 (1X 10 each) ⁴ Individual cells) were cultured in triplicate. After 16 hours, the top 150 μ l of medium was transferred to a V-bottom 96-well plate and centrifuged at 300g for 5 minutes to pellet any remaining cells. In some experiments, in E: t =10:1, the supernatant was used for cytokine ELISA assay. Supernatants were transferred to new 96-well plates and human cytokine levels (IFN-. Gamma., IL-2, IL-6) were analyzed by ELISA using a kit from Sammer Feishale (south san Francisco, CA) according to the manufacturer's protocol.

Example 8 growth of mouse xenograft tumors

6-week-old Male NSG mice (Jack-mice) were treated strictly according to the institutional review Board for animal protection and use (IACUC)Son laboratory, port of balk, ME) for feeding and handling. Each mouse was injected subcutaneously with 2X 10 in 100. Mu.l sterile PBS ⁶ BxPC3 cells, followed by intratumoral injection of CAR-T cells three times: administration was 2X 10 on days 20, 27 and 34, respectively ⁵ Cell/mouse, 2X 10 ⁶ Cell/mouse, 2.8X 10 ⁶ Cell/mouse dose, and analysis of xenograft tumor growth. Tumor size was measured twice weekly with calipers and using the formula W ² L/2 determination of tumor volume (mm) ³ ) Where W is the tumor width and L is the tumor length. Tumors were excised and fixed in 4% paraformaldehyde, then embedded in paraffin and stained by immunohistochemistry. At the end of the intravenous CAR-T cell study, 100 μ L of blood was collected and stained with different antibodies by flow cytometry as described above.

Example 9 Immunohistochemical (IHC) staining

Tumor tissue sections (4 μm) were incubated twice in xylene for 10 min, then hydrated in gradient alcohol and rinsed in PBS. Antigen retrieval was performed in an autoclave for 20 minutes using 10mM citrate buffer (pH 6.0). The sections were cooled, rinsed with PBS, and rinsed at 3%H ₂ O ₂ Incubate in solution for 10 minutes and rinse with PBS. Tissue sections were incubated in goat serum for 20 minutes and then overnight at 4 ℃ with 0.2. Mu.g/ml rabbit anti-lytic caspase-3 (Asp 175, cell signaling technology, denfoss, MA, US) or rabbit IgG (Jackson immuno Studies, west Grov, PA, USA). Sections were rinsed with PBS, incubated with biotin-conjugated goat anti-rabbit IgG for 10 min, rinsed with PBS, incubated with streptavidin-conjugated peroxidase for 10 min, and rinsed with PBS. Finally, sections were incubated in DAB matrix solution for 2-5 minutes, immersed in tap water, counterstained with hematoxylin, rinsed with water, and dehydrated in gradient alcohol and xylene. The coverslip was mounted with glycerol. Images were obtained on a DMB5-2231PL microscope (Motic, xiamen, china) using Images Plus 2.0 software.

Example 10 humanization of CD47 antibodies

Mouse B6H12 CDRs were humanized using IgBLAST (NCBI) software according to the method described by Almagro et al (front. Biosci.,2008,13,1619-1633). The human framework of the human clone with the highest homology was used for humanization pairing. Mouse CDRs were inserted into these clones, yielding four different variants, which were tested by ELISA.

Example 11 binding assay of humanized and mouse CD47scFv

The mouse or humanized CD47ScFv contains VL and VH sequences linked to a G4Sx3 linker. ScFv was fused in-frame to human Fc in pYD11 vector for recombinant CD47ScFv protein expression via C-terminus. An equal amount of supernatant containing mammalian cells expressing ScFv protein was used for the binding assay. Expression of all ScFv was checked by Western blotting using anti-human Fc antibody. The human extracellular domain (19-41 amino acids) of the human CD47 protein (Gen Bank ID: NM-001777.3) was fused to mouse Fc and used for ELISA assays for CD47ScFv. OD readings at 450nm were used to detect binding. Based on the mouse sequences, computer simulation models were generated for the humanized VH and VL sequences.

As a result, the

Statistical analysis

Data were analyzed and plotted using Prism software (GraphPad, san diego, CA). Comparisons between the two groups were made by unpaired student's t-test, and between the three groups by one-way analysis of variance and Tukey's post hoc test, unless otherwise noted. Differences were considered significant at p-values <0.05.

Example 12 sequence of mouse CD47ScFv CAR construct

The amino acid sequences of all segments of the CD47CAR constructs used in the experiments are shown below. Each segment may be replaced with an amino acid sequence having at least 95% identity.

< human CD8 signal peptide > SEQ ID NO:2

M A L P V T A L L L P L A L L L H A A R P

< Nhe I site > this site can optionally be used to excise scFv if desired, and can be replaced by other restriction enzyme sites.

AS

Mouse anti-CD 47scFv (VH-linker-VL)

<VH>SEQ ID NO:3

E V Q L V E S G G D L V K P G G S L K L S C A A S G F T F S G Y G M S W V R Q T P D K R L E W V A T I T S G G T Y T Y Y P D S V K G R F T I S R D N A K N T L Y L Q I D S L K S E D T A I Y F C A R S L A G N A M D Y W G Q G T S V T V S S

< linker > SEQ ID NO 4

GGGGSGGGGSGGGGS

<VL>SEQ ID NO:5

D I V M T Q S P A T L S V T P G D R V S L S C R A S Q T I S D Y L H W Y Q Q K S H E S P R L L I K F A S Q S I S G I P S R F S G S G S G S D F T L S I N S V E P E D V G V Y Y C Q N G H G F P R T F G G G T K L E I K

< Xho I site > this site can optionally be used to excise scFv if necessary, and can be replaced by other restriction enzyme sites.

LE

< CD8 hinge > SEQ ID NO:6

K P T T T P A P R P P T P A P T I A S Q P L S L R P E A S R P A A G G A V H T R G L D F A S D K P

< transmembrane domain TM28> SEQ ID NO:7

F W V L V V V G G V L A C Y S L L V T V A F I I F W V

< co-stimulatory domain CD28> SEQ ID NO:8

R S K R S R L L H S D Y M N M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S

< activation domain CD3- ζ > SEQ ID NO:9

R V K F S R S A D A P A Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D P E M G G K P R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R

The mouse CD47ScFv CAR sequence is set forth in SEQ ID NO:10, and:

M A L P V T A L L L P L A L L L H A A R P A S E V Q L V E S G G D L V K P G G S L K L S C A A S G F T F S G Y G M S W V R Q T P D K R L E W V A T I T S G G T Y T Y Y P D S V K G R F T I S R D N A K N T L Y L Q I D S L K S E D T A I Y F C A R S L A G N A M D Y W G Q G T S V T V S S G G G G S G G G G S G G G G S D I V M T Q S P A T L S V T P G D R V S L S C R A S Q T I S D Y L H W Y Q Q K S H E S P R L L I K F A S Q S I S G I P S R F S G S G S G S D F T L S I N S V E P E D V G V Y Y C Q N G H G F P R T F G G G T K L E I K L E K P T T T P A P R P P T P A P T I A S Q P L S L R P E A S R P A A G G A V H T R G L D F A S D K P F W V L V V V G G V L A C Y S L L V T V A F I I F W V R S K R S R L L H S D Y M N M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S R V K F S R S A D A P A Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D P E M G G K P Q R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R

in the same way, "mock" CARs can be constructed which contain scfvs specific for intracellular proteins and thus do not react with intact cells-.

Example 13 sequences of humanized CD47ScFv CAR constructs

The VH and VL of the mouse CD47 antibody B6H12 were humanized using bioinformatics Methods according to the Methods described by Almagro et al (front. Biosci.2008,13, 1619-1633) and Gilliland et al (Methods mol. Biol.,2012,841, 321-349).

In our experiments, the amino acid sequences of all segments of the humanized CD47ScFv CAR construct used were identical to those shown in example 12, except that the sequence of the mouse anti-CD 47ScFv was replaced by a humanized anti-CD 47 sequence as shown below. Each segment may be replaced with an amino acid sequence having at least 95% sequence identity.

Humanized anti-CD 47scFv (VH-linker-VL)

<VH>SEQ ID NO:11

EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYGMSWVRQAPGKGLEWVATITSGGTYTYYPDSVKGRFTISRDNA KNSLYLQMNSLRAEDTAVYYCARSLAGNAMDYWGQGTLVTVSS

< linker > SEQ ID NO 4

GGGGSGGGGSGGGGS

<VL>SEQ ID NO:12

EIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYFASQRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGHGFPRTFGGGTKVEIK

The humanized CD47scFv is shown as SEQ ID No. 13:

E V Q L V E S G G G L V Q P G G S L R L S C A A S G F T F S G Y G M S W V R Q A P G K G L E W V A T I T S G G T Y T Y Y P D S V K G R F T I S R D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R S L A G N A M D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S E I V L T Q S P A T L S L S P G E R A T L S C R A S Q S I S D Y L H W Y Q Q K P G Q A P R L L I Y F A S Q R A T G I P A R F S G S G S G T D F T L T I S S L E P E D F A V Y Y C Q Q G H G F P R T F G G G T K V E I K

humanized CD47ScFv CAR is shown in SEQ ID NO:14, in the following:

M A L P V T A L L L P L A L L L H A A R P A S E V Q L V E S G G G L V Q P G G S L R L S C A A S G F T F S G Y G M S W V R Q A P G K G L E W V A T I T S G G T Y T Y Y P D S V K G R F T I S R D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R S L A G N A M D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S E I V L T Q S P A T L S L S P G E R A T L S C R A S Q S I S D Y L H W Y Q Q K P G Q A P R L L I Y F A S Q R A T G I P A R F S G S G S G T D F T L T I S S L E P E D F A V Y Y C Q Q G H G F P R T F G G G T K V E I K L E K P T T T P A P R P P T P A P T I A S Q P L S L R P E A S R P A A G G A V H T R G L D F A S D K P F W V L V V V G G V L A C Y S L L V T V A F I I F W V R S K R S R L L H S D Y M N M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S R V K F S R S A D A P A Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D P E M G G K P Q R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R

example 14 bispecific EGFR-Flag-CD47-CD28-CD3CAR construct

In our experiments, the amino acid sequence of each segment of the EGFR-Flag-CD47 ScFv CAR construct used was the same as shown in example 12, except that the EGFR ScFv, flag and linker sequences shown below were added at the N-terminus of the mouse anti-CD 47 sequence. Each segment may be replaced with an amino acid sequence having at least 95% sequence identity.

Human anti-EGFR scFv (VH-linker-VL-FLAG-linker)

<VH>SEQ ID NO:15

E V Q L V Q S G A E V K K P G S S V K V S C K A S G G T F S S Y A I S W V R Q A P G Q G L E W M G G I I P I F G T A N Y A Q K F Q G R V T I T A D E S T S T A Y M E L S S L R S E D T A V Y Y C A R E E G P Y C S S T S C Y G A F D I W G Q G T L V T V S S

< linker > SEQ ID NO 4

GGGGSGGGGSGGGGS

<VL>SEQ ID NO:16

Q S V L T Q D P A V S V A L G Q T V K I T C Q G D S L R S Y F A S W Y Q Q K P G Q A P T L V M Y A R N D R P A G V P D R F S G S K S G T S A S L A I S G L Q S E D E A D Y Y C A A W D D S L N G Y L F G A G T K L T V L

< FLAG > SEQ ID NO:1,FLAG tag is optional and is used to detect expression.

< linker > SEQ ID NO 4

The sequence of bispecific EGFR-CD47 ScFv CAR is shown in SEQ ID NO:17 shows:

M A L P V T A L L L P L A L L L H A A R P A S E V Q L V Q S G A E V K K P G S S V K V S C K A S G G T F S S Y A I S W V R Q A P G Q G L E W M G G I I P I F G T A N Y A Q K F Q G R V T I T A D E S T S T A Y ME L S S L R S E D T A V Y Y C A R E E G P Y C S S T S C Y G A F D I W G Q G T L V T V S S G G G G S G G G G S G G G G S Q S V L T Q D P A V S V A L G Q T V K I T C Q G D S L R S Y F A S W Y Q Q K P G Q A P T L V M Y A R N D R P A G V P D R F S G S K S G T S A S L A I S G L Q S E D E A D Y Y C A A W D D S L N G Y L F G A G T K L T V L D Y K D D D D K G G G G S G G G G S G G G G S D I V M T Q S P A T L S V T P G D R V S L S C R A S Q T I S D Y L H W Y Q Q K S H E S P R L L I K F A S Q S I S G I P S R F S G S G S G S D F T L S I N S V E P E D V G V Y Y C Q N G H G F P R T F G G G T K L E I K G G G G S G G G G S G G G G S E V Q L V E S G G D L V K P G G S L K L S C A A S G F T F S G Y G M S W V R Q T P D K R L E W V A T I T S G G T Y T Y Y P D S V K G R F T I S R D N A K N T L Y L Q I D S L K S E D T A I Y F C A R S L A G N A M D Y W G Q G T S V T V S S L E K P T T T P A P R P P T P A P T I A S Q P L S L R P E A S R P A A G G A V H T R G L D F A S D K P F W V L V V V G G V L A C Y S L L V T V A F I I F W V R S K R S R L L H S D Y M N M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S R V K F S R S A D A P A Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D P E M G G K P Q R R K N P Q E G L Y N E L Q K D K MA E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R

example 15 killing of CD47-CAR-T cells by mouse CD 47-Positive cancer cells

The CD47ScFv was used to generate CD47-CAR-T cells (second generation CAR-T cells) with a CD28 costimulatory domain and a CD zeta activating domain (figure 2B). At day 14, CD47-CAR-T cells were expanded > 150-fold in vitro and CAR expression was confirmed by F (ab) 2 antibody FACS. To detect killing activity in vitro, CD47 positive cells expressing high levels of CD47 antigen were used by RTCA assay (real-time cytotoxicity assay): ovarian cancer cells, A1847 and SKOV-3, as well as cancer cells expressing low levels of CD47, A549 cells and Hep3B cells, CD47CAR-T cells were tested. CD47-CAR-T cells kill high expressing cancer cell lines such as a1847, SKOV-3 efficiently, and CAR-T cells kill less cells of a549, hep3B, and low levels of CD47 than T cells or Mock-CAR-T cells. The cytotoxic effect of cancer cells was significantly higher than T cells and Mock-CAR-T cells, p <0.0001 (fig. 3A). This demonstrates the CD 47-dependent activity of CD47-CAR-T cells depending on the expression of the CD47 antigen.

CD47-CAR-T cells produced significantly higher IL-2 cytokines against cancer cells in SKOV3 cells that were highly positive for CD47 compared to a549 and Hep3B cells with low expression of CD47 (fig. 3B). Thus, based on CD47 expression on the surface of cancer cells, CD47-CAR-T cells kill and secrete IL-2 cytokines in a CD 47-dependent manner, consistent with the cytotoxicity data.

Example 16 CD47-CAR-T cells significantly reduce BxPC3 pancreatic cancer xenograft tumor growth

To test the in vivo efficacy of CD47-CAR-T cells, we used BxPC3 pancreatic cancer cells. Cytotoxicity of CD47-CAR-T cells was compared to Mock CAR-T control cells and CD24-CAR-T cells. Figures 4A-4G show that CD47-CAR-T cells significantly inhibited the growth of BxPC3 pancreatic cancer xenograft tumors. CD24-CAR-T cells with CD24-CAR ScFv were used as non-CD 47 control CAR-T cells based on BxPC3 cells with significantly lower CD24 expression compared to CD47 (fig. 4A). CD47-CAR-T cells showed high cytotoxicity to BxPC3 cells compared to Mock control CAR-T cells and CD24-CAR-T cells (fig. 4B).

Furthermore, CD47-CAR-T cells expressed significantly higher levels of cytokines compared to Mock and CD24-CAR-T cells: IFN-. Gamma.IL-2 (FIG. 4C); and, compared to Mock control and CD24-CAR-T cells, expressed high levels of IL-6 against target BxPC3 cells (fig. 4C). BxPC3 cancer cells were injected subcutaneously into NSG mice to generate the constructed xenograft tumors (fig. 4D). Subsequently, three injections, CD47-CAR-T cells, control 1 × PBS and CD24-CAR-T cells, were administered intratumorally on days 20, 27 and 34. CD47-CAR-T cells significantly inhibited the growth of BxPC3 xenograft tumors compared to controls, with p <0.05 (fig. 4D). CD47-CAR-T cells did not affect mouse body weight (fig. 4E). The tumor size (figure 4F) and weight (figure 4G) from the CD47-CAR-T cell treated group was significantly lower (p < 0.05) than the control 1x PBS and CD24-CAR-T cell groups.

Example 17 efficient binding of humanized CD47ScFv to CD47 antigen and detection of CD47 in tumor samples

A humanized version of the CD47ScFv with high affinity for the CD47 antigen is shown in figure 5A. The humanized VH and the mouse B6H12VH have the same CDR1,2,3 regions (CDR regions in italics and underlined). The humanized VH and the mouse B6H12VH have similar human framework regions, with differences in the framework regions underlined in larger font. (FIG. 5A)

As for VL, the VL of the humanized form of the CD47 antibody had three amino acid changes in CDR2 and one in the CDR3 region compared to the mouse B6H12 (CDRs are all underlined and shown in italics, changes in CDRs are shown in larger font and bold; CDR3 region is important for antigen binding) (FIG. 5A).

Binding of the humanized CD47ScFv was tested by ELISA (fig. 5B). It had significantly higher binding than the mouse CD47ScFv and higher binding than the negative control PDL-1 (fig. 5B). For detection of CD47 antigen, humanized CD47ScFv could be better detected in lymphoma samples, similar in other tumor samples (ovarian cancer, gastric cancer (not shown)), and less or similarly detected in normal tissues such as tonsils (fig. 5C).

Example 18 humanized CD47-CAR-T cells effectively kill cancer cells in a CD 47-dependent manner

Against CD47 high expressing ovarian cancer cell lines: SKOV-3, a1847 and pancreatic cancer cell line BxPC3, as well as low-expressing Hela-CD19 cancer cell line, humanized CD47-CAR T cells were detected (fig. 6A). The humanized CD47-CAR-T cells efficiently killed CD47 positive cancer cells, had no killing activity on Hela-CD19 cancer cells with very low CD47 expression, while the positive control CD19-CAR-T cells efficiently killed Hela-CD19 cells (fig. 6A, bottom right panel). The cytotoxicity of CD47-CAR-T cells on CD47 positive cells was significantly increased (p < 0.05) compared to CD47 negative cells and control Mock CAR-T cells (fig. 6B). The level of IL-2 cytokines secreted by CD47-CAR-T cells was also significantly increased (p < 0.05) in SKOV-3-CD47 positive cell lines compared to Mock-CAR-T cells (fig. 6C), but absent in CD47 negative Hela-CD19 cells (fig. 6D). In contrast, control CD19-CAR-T cells produced significantly increased levels of IL-2 (p < 0.05) in Hela-CD19 positive cells compared to Mock-CAR-T cells (fig. 6D). Thus, humanized CD47-CAR-T cells effectively and specifically kill cancer cell lines and produce cytokines in a CD 47-dependent manner.

Example 19 bispecific EGFR-CD47CAR-T cell killing of cancer cells

The EGFR (C10) human antibody scFv-FLAG was ligated to CD47scFv by using a G4Sx3 linker to generate a bispecific CAR. (see example 14).

Bispecific EGFR-CD47CAR-T cells killed EGFR-negative MCF-7 cells, exhibiting CD 47-specific activity (fig. 7). In addition, EGFR-CD47CAR-T cells efficiently killed EGFR-positive and CD 47-positive BxPC3 pancreatic cancer cells (fig. 8A) and SKOV ovarian cancer cells (fig. 8B). These data demonstrate the utility of a bispecific EGFR-CD47 CAR.

It will be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made therein without departing from the scope of the invention as set forth in the claims.

Sequence listing

<110> Priman Biotechnology Ltd

PROMAB BIOTECHNOLOGIES, Inc.

<120> CD47-CAR-T cells

<130> P2018-1564

<150> US 62/458,773

<151> 2017-02-14

<150> US 62/518,767

<151> 2017-06-13

<150> US 62/546,790

<151> 2017-08-17

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 2

<211> 21

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 3

<211> 118

<212> PRT

<213> mouse (Mus musculus)

<400> 3

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Ile Asp Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Phe Cys

85 90 95

Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 4

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 5

<211> 107

<212> PRT

<213> mouse (Mus musculus)

<400> 5

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Gly Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 6

<211> 49

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 6

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys

35 40 45

Pro

<210> 7

<211> 27

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 7

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 8

<211> 41

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 8

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 9

<211> 112

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 9

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 10

<211> 495

<212> PRT

<213> mouse (Mus musculus)

<400> 10

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ala Ser Glu Val Gln Leu Val Glu Ser Gly Gly

20 25 30

Asp Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser

35 40 45

Gly Phe Thr Phe Ser Gly Tyr Gly Met Ser Trp Val Arg Gln Thr Pro

50 55 60

Asp Lys Arg Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Thr Tyr

65 70 75 80

Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

85 90 95

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Ile Asp Ser Leu Lys Ser Glu

100 105 110

Asp Thr Ala Ile Tyr Phe Cys Ala Arg Ser Leu Ala Gly Asn Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser

165 170 175

Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr Leu His Trp Tyr

180 185 190

Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Phe Ala Ser

195 200 205

Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly

225 230 235 240

Val Tyr Tyr Cys Gln Asn Gly His Gly Phe Pro Arg Thr Phe Gly Gly

245 250 255

Gly Thr Lys Leu Glu Ile Lys Leu Glu Lys Pro Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

370 375 380

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

385 390 395 400

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

405 410 415

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 11

<211> 118

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 12

<211> 107

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 12

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Phe Ala Ser Gln Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly His Gly Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 240

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu

130 135 140

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

145 150 155 160

Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

165 170 175

Pro Arg Leu Leu Ile Tyr Phe Ala Ser Gln Arg Ala Thr Gly Ile Pro

180 185 190

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

195 200 205

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly

210 215 220

His Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

225 230 235 240

<210> 14

<211> 495

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 14

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ala Ser Glu Val Gln Leu Val Glu Ser Gly Gly

20 25 30

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

35 40 45

Gly Phe Thr Phe Ser Gly Tyr Gly Met Ser Trp Val Arg Gln Ala Pro

50 55 60

Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Thr Tyr

65 70 75 80

Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

85 90 95

Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Leu Ala Gly Asn Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Phe Ala Ser

195 200 205

Gln Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Tyr Cys Gln Gln Gly His Gly Phe Pro Arg Thr Phe Gly Gly

245 250 255

Gly Thr Lys Val Glu Ile Lys Leu Glu Lys Pro Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

370 375 380

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

385 390 395 400

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

405 410 415

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 15

<211> 126

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 15

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Glu Gly Pro Tyr Cys Ser Ser Thr Ser Cys Tyr Gly Ala

100 105 110

Phe Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 16

<211> 108

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 16

Gln Ser Val Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Lys Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Phe Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Thr Leu Val Met Tyr

35 40 45

Ala Arg Asn Asp Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly

85 90 95

Tyr Leu Phe Gly Ala Gly Thr Lys Leu Thr Val Leu

100 105

<210> 17

<211> 767

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ala Ser Glu Val Gln Leu Val Gln Ser Gly Ala

20 25 30

Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser

35 40 45

Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro

50 55 60

Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr

65 70 75 80

Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp

85 90 95

Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Glu Gly Pro Tyr Cys Ser

115 120 125

Ser Thr Ser Cys Tyr Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu

130 135 140

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Asp Pro Ala Val Ser Val

165 170 175

Ala Leu Gly Gln Thr Val Lys Ile Thr Cys Gln Gly Asp Ser Leu Arg

180 185 190

Ser Tyr Phe Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Thr

195 200 205

Leu Val Met Tyr Ala Arg Asn Asp Arg Pro Ala Gly Val Pro Asp Arg

210 215 220

Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly

225 230 235 240

Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp

245 250 255

Ser Leu Asn Gly Tyr Leu Phe Gly Ala Gly Thr Lys Leu Thr Val Leu

260 265 270

Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala

290 295 300

Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala

305 310 315 320

Ser Gln Thr Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His

325 330 335

Glu Ser Pro Arg Leu Leu Ile Lys Phe Ala Ser Gln Ser Ile Ser Gly

340 345 350

Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu

355 360 365

Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln

370 375 380

Asn Gly His Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu

385 390 395 400

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

405 410 415

Ser Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly

420 425 430

Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly

435 440 445

Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp

450 455 460

Val Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser

465 470 475 480

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

485 490 495

Tyr Leu Gln Ile Asp Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Phe

500 505 510

Cys Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly

515 520 525

Thr Ser Val Thr Val Ser Ser Leu Glu Lys Pro Thr Thr Thr Pro Ala

530 535 540

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

545 550 555 560

Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr

565 570 575

Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Phe Trp Val Leu Val Val

580 585 590

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

595 600 605

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

610 615 620

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

625 630 635 640

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

645 650 655

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

660 665 670

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

675 680 685

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln

690 695 700

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

705 710 715 720

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

725 730 735

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

740 745 750

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

755 760 765

Claims

1. A chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus:

(i) Comprising V _H And V _L The single chain variable fragment (scFv) of (1), wherein the scFv is directed against the CD47 tumor antigen,

(ii) (ii) a transmembrane domain which is capable of,

(iii) (ii) a CD28 co-stimulatory domain,

(iv) An activation domain, wherein said scFv is derived from a humanized CD47 antibody and comprises the amino acid sequence of SEQ ID NO:11 and 12.

2. The fusion protein of claim 1, wherein the humanized scFv has the amino acid sequence of SEQ ID NO:13, or a pharmaceutically acceptable salt thereof.

3. The fusion protein of claim 1, wherein the costimulatory domain is selected from the group consisting of: CD28, 4-1BB, ICOS-1, CD27, OX-40, DAP10, and GITR.

4. The fusion protein of claim 1, wherein the costimulatory domain is CD28.

5. The fusion protein of claim 1, wherein the activation domain is CD3 ζ.

6. The fusion protein of claim 1, wherein the fusion protein has the amino acid sequence of SEQ ID No:14, or a pharmaceutically acceptable salt thereof.

7. The fusion protein of claim 1, wherein (i) further comprises a second single chain variable fragment (scFv) comprising a second V _H And a second V _L And the second scFv is directed against an EGFR tumor antigen.

8. A nucleic acid sequence encoding the fusion protein of any one of claims 1-7.

9. A cell modified to express the fusion protein of any one of claims 1-7.